Cloning and expression of murine immune interferon cDNA

Area of Science:

• Molecular Biology
• Immunology
• Gene Expression

Background:

• Interferon-gamma (IFN-gamma) is a critical cytokine involved in immune responses.
• Understanding the genetic basis and expression of IFN-gamma across species aids in comparative immunology and therapeutic development.

Purpose of the Study:

• To clone and express the murine immune interferon-gamma (IFN-gamma) gene.
• To characterize the biological, antigenic, and biochemical properties of the expressed murine IFN-gamma.
• To compare the murine IFN-gamma gene and protein with its human counterpart.

Main Methods:

• Cloning of the murine IFN-gamma gene into an expression vector.
• Transfection and expression in monkey COS-1 cell line under simian virus 40 early promoter control.
• RNA isolation from mitogen-induced murine spleens and transfected COS cells for cDNA cloning.
• Expression of murine IFN-gamma cDNA in Escherichia coli under trp promoter control.

Main Results:

• A functional murine IFN-gamma protein, identical to the natural form, was secreted from transfected COS cells.
• The mature murine IFN-gamma protein consists of 136 amino acids, 10 fewer than human IFN-gamma.
• Nucleotide homology between murine and human IFN-gamma genes is 60-65%, with only 40% homology at the protein level.
• Successful expression of murine IFN-gamma cDNA in Escherichia coli was achieved.

Conclusions:

• The murine IFN-gamma gene can be successfully cloned and expressed in a heterologous mammalian system (COS cells).
• The expressed murine IFN-gamma protein exhibits authentic biological, antigenic, and biochemical characteristics.
• Significant sequence divergence exists between murine and human IFN-gamma at both nucleotide and amino acid levels.